Materials and methods useful to affect growth and development of lepidoptera larvae

ABSTRACT

The present invention provides materials and methods related to causing insect resistance in plants. Specifically, the present invention provides nucleic acid compounds which encode a teratocyte secreted protein (TSP) which is capable of causing inhibition of growth and developmental arrest in Lepidoptera larvae. Cells and plants (including plant parts, seeds, embryos, etc.) comprising the nucleic acid compounds are also within the scope of the present invention. The present invention also provides amino acid compounds encoded by the nucleic acid compounds of the present invention, as well as methods to induce larval developmental arrest, methods to produce the nucleic acid and amino acid compounds, and methods to inhibit crop damage due to Lepidoptera infestation.

BACKGROUND OF THE INVENTION

Parasites of plant-eating insects are natural pesticides. One suchsolitary parasite, Microplitis croceipes (a member of the wasp familyBraconidae), causes inhibition of growth and permanent developmentalarrest of the tobacco budworm, the cotton bollworm (also known as thecorn earworm and tomato fruitworm) and the soybean pod worm. The larvaeof these insects (Heliothis and Helicoverpa spp.) feed on tobacco,cotton, maize, sorghum, soybeans, sunflower and tomatoes, among otherplants. The larvae cause economic losses of over $1 billion annually,primarily in the form of yield reduction and costs related to control.Fitt, 34 Ann. Rev. Entomol. 17 (1989).

In the past, the United States Department of Agriculture has fundedprograms designed to increase Microplitis croceipes populations inSouthern states so as to naturally combat these pests in those areas.These efforts were successful in reducing pest populations. Use of such“biomanagement” techniques has generally been replaced with molecularbiology techniques, such as those which allowed engineering of plantswhich express Bacillus thuringiensis toxin.

Certain extra-embryonic cells (teratocytes) from M. croceipes have beenshown to be involved in impairment of the growth, development andrelated physiological parameters of Heliothis and Helicoverpa larvae.Although teratocytes do not undergo cell division subsequent to theirrelease into the hemocoel of the host, they do become polyploidal.Injection of one larval equivalent of teratocytes caused characteristicpost-wandering, pre-pupation developmental arrest and eventual deathassociated with parasitization. Zhang and Dahlman, 12 Arch. InsectBiochem. Physiol. 51 (1989). Teratocytes collected from M. croceipeseggs hatched in vitro produced responses similar to those collected fromparasitized Heliothis larvae. Zhang et al., 43 J. Insect Physiol. 577(1997). Furthermore, it has been suggested that when teratocytes arecultured in vitro, they secrete a mixture of proteins (teratocytesecreted proteins or TSP) which, when injected into host larvae,produced responses similar to parasitization. Schepers et al., 44 J.Insect Physiol. 767 (1998).

In related studies, juvenile hormone esterase and ecdysone titers havebeen shown to be suppressed by teratocytes to a degree similar to thosein parasitized larvae. Zhang, et al. 20, Arch. Insect Biochem. Physiol.231 (1992). Reduced titers of host hemolymph proteins have beenobserved, particularly in older stages, with a major effect on the 74,76 and 82 kD storage protein monomers. Zhang et al., 43 J. InsectPhysiol. 577 (1997). Inhibition of storage protein synthesis in the fatbody was theorized to be at the level of translation, primarily based onthe finding that storage protein mRNA levels did not change, even thoughprotein synthesis declined precipitously after treatment withteratocytes. Dong et al., 32 Arch. Insect Biochem. Physiol. 237 (1996).

Citation of the above documents is not intended as an admission that anyof the foregoing is pertinent prior art. All statements as to the dateor representation as to the contents of these documents is based onsubjective characterization of information available to the applicant,and does not constitute any admission as to the accuracy of the dates orcontents of these documents.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide isolatednucleic acid compounds useful to inhibit insect larval growth anddevelopment.

It is a further object to provide isolated amino acid compounds usefulto inhibit larval growth and development.

It is yet another object to provide methods to produce isolated aminoacid compounds useful to inhibit larval growth and development.

It is yet another object to provide methods to inhibit larval growth anddevelopment.

In all of the above embodiments, it is an object to provide methods toreduce crop damage using the compounds and methods herein.

It is an object of the invention to provide a natural pesticide usingthe compounds and methods herein.

It is an object of the invention to provide vectors, cells and molecularconstructs comprising the compounds and methods herein.

It is an object of the invention to provide plants, including plantparts, seeds and embryos comprising the compounds and methods herein.

Definitions:

For the purposes of the present application, the following terms havethe following meanings. All other terms have the meaning as generallyrecognized in the art.

“Allelic variant” is meant to refer to a gene that occurs at essentiallythe same locus (or loci) as the referent sequence, but which, due tonatural variations caused by, for example, mutation or recombination,has a similar but not identical sequence. Allelic variants typicallyencode proteins having similar activity to that of the protein encodedby the gene to which they are being compared. Allelic variants can alsocomprise alterations in the 5′ or 3′ untranslated regions of the gene(e.g., in regulatory control regions).

“Fragment” is meant to refer to any nucleic acid or polypeptide subsetof the referent compound.

“Inducing agent” means any compound or condition that causes inducementof an inducible promoter, including chemical compounds or environmentalconditions, such as drought, wounds, light cycle, etc.

“Maize” and “corn” shall be interchangeable and mean all maizevarieties.

“Proteins” means any compounds which comprise amino acids, includingpeptides, polypeptides, fusion proteins, etc.

“Transform” means delivery of nucleic acid into a cell, includingdelivery which results in genomic integration and delivery which resultsin transient localization within the cell membrane.

Moreover, for the purposes of the present invention, the term “a” or“an” entity refers to one or more of that entity; for example, “aprotein” or “a nucleic acid molecule” refers to one or more of thosecompounds or at least one compound. As such, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising”, “including”, and“having” can be used interchangeably. Furthermore, a compound “selectedfrom the group consisting of” refers to one or more of the compounds inthe list that follows, including mixtures (i.e., combinations) of two ormore of the compounds. According to the present invention, an isolated,or biologically pure, protein or nucleic acid molecule is a compoundthat has been removed from its natural milieu. As such, “isolated” and“biologically pure” do not necessarily reflect the extent to which thecompound has been purified. An isolated compound of the presentinvention can be obtained from its natural source, can be produced usingmolecular biology techniques or can be produced by chemical synthesis.

BRIEF DESCRIPTION OF THE TABLES

Table 1—Sequence listing of cDNAs, peptides and primers.

Table 2—Responses of Heliothis virescens larvae to feeding on transgenicplants expressing the TSP gene.

Table 3—Responses of Manduca sexta larvae to feeding on transgenicplants expressing the TSP gene.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that when the recombinant TSP was tested with a rabbitreticulocyte lysate assay an inverse dose-dependent effect was observedover a range of 0.2-0.8 μg protein/25 μL, showing that TSP inhibitsprotein synthesis.

FIG. 2 shows a schematic map of the plant expression vectors constructedfor expressing the chimeric TSP gene with its own signal peptide(pKT117) and without any signal peptide (pKT118); and with signalpeptide from the PR1b protein (pKT119), showing the coding sequencedirected by the peanut chlorotic streak virus full-length transcriptpromoter (2×enh PFLt), one modified with double enhancer domains [Maitiand Shepherd 244, Biochem. Biophys. Res. Comm. 440 (1998); Maiti andShepherd, U.S. Pat. No., 5,850,019.

FIG. 3 shows expression analysis of TSP genes in different transgenicplant lines. The RNA Dot Blot shows negative response to control andvector controls but varying levels of RNA in 8 different transgenicplant lines. The Northern Blot shows presence of TSP RNA in lanes 4, 5,and 8. Western Blot analysis contains TSP control in lane 1, controlleaf extract in lane 2, control seedling extract in lane 3, transformedleaf extract in lane 4 and transformed seedling extract in lane 5.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides materials and methods related to causinggrowth inhibition and developmental arrest of Lepidoptera larvae.

Specifically, the present invention provides nucleic acid compoundswhich encode a teratocyte secreted protein (named TSP for the purposesof the present invention) which is capable of causing inhibition ofgrowth and developmental arrest of Heliothis larvae. A nucleic acidcompound specifically set forth in the sequence listing encodes one suchteratocyte secreted protein from M. croceipes. The nucleic acidcompounds include fragments and complements of the sequence in thesequence listing, as well as sequences which can be obtained, withoutundue experimentation, by virtue of the knowledge of these sequences.The nucleic acid compounds herein provided therefore include variationson the fragments, complements and other sequences, such as vectors orother constructs containing the fragments, complements, allelic variantsand homologues. Cells and plants (including plant parts, seeds, embryos,etc.) comprising the nucleic acid compounds are also within the scope ofthe present invention.

The present invention also provides amino acid compounds encoded by thenucleic acid compounds of the present invention, as well as methods toinduce larval developmental arrest, methods to produce the nucleic acidand amino acid compounds, and methods to reduce crop damage due to avariety of Lepidopteran larval pests, including Heliothis andHelicoverpa species infestations.

Therefore, the present invention provides isolated nucleic acidmolecules encoding a TSP protein molecule, wherein said nucleic acidmolecule comprises a nucleic acid sequence selected from the groupconsisting of:

(a) a nucleic acid molecule which has more than 70% identity to anucleic acid molecule selected from the group consisting of: SEQ ID NO 1and SEQ ID NO 3, wherein said identity can be determined using theDNAsis computer program and default parameters;

(b) a nucleic acid molecule which encodes an amino acid sequence whichcomprises an amino acid sequence which has more than 70% identity to anamino acid sequence selected from the group consisting of: SEQ ID NO 2and SEQ ID NO 4, wherein said identity can be determined using theDNAsis computer program and default parameters;

(c) a nucleic acid molecule which is an allelic variant of a nucleicacid molecule selected from the group consisting of: a nucleic acidmolecule of (a); and a nucleic acid molecule of (b); and

(d) a nucleic acid molecule fully complementary to a nucleic acidmolecule selected from the group consisting of: a nucleic acid moleculeof (a); a nucleic acid molecule of (b); and a nucleic acid molecule of(c).

Also provided are vectors, in particular vectors comprising induciblepromoters, and for those comprising inducible promoters, in particularthose which are tightly regulated. Recombinant plant cells, seeds, plantparts, embryos and plants comprising these vectors are also provided.Preferred are plants selected from the group consisting of tobacco,cotton, corn, soybeans, and tomatoes.

It is well-known in the art that there are commercially availablecomputer programs for determining the degree of similarity between twonucleic acid sequences. These computer programs include various knownmethods to determine the percentage identity and the number and lengthof gaps between hybrid nucleic acid molecules. Preferred methods todetermine the percent identity among amino acid sequences and also amongnucleic acid sequences include analysis using one or more of thecommercially available computer programs designed to compare and analyzenucleic acid or amino acid sequences. These computer programs include,but are not limited to, GCG^(a) (available from Genetics Computer Group,Madison, Wis.), DNAsis^(a) (available from Hitachi Software, San Bruno,Calif.) and MacVector^(a) (available from the Eastman Kodak Company, NewHaven, Conn.). A preferred method to determine percent identity amongamino acid sequences and also among nucleic acid sequences includesusing the Compare function by maximum matching within the program DNAsisVersion 2.1 using default parameters. A nucleic acid sequence of thepresent invention may have at least 65%, preferably 70%, and mostpreferably 90% sequence identity with a nucleic acid molecule in thesequence listing. However, any percent identity within the range of 65through 100% is within the scope of the present invention. Therefore,molecules with 80%, 85% and 95% identity are also within the scope ofthe present invention.

Therefore, also provided are isolated nucleic acid compounds encoding aTSP protein molecule, wherein said nucleic acid compound is selectedfrom the group consisting of:

(a) a nucleic acid molecule which has more than 90% identity to anucleic acid molecule selected from the group consisting of: SEQ ID NO 1and SEQ ID NO 3, wherein said identity can be determined using theDNAsis computer program and default parameters

(b) a nucleic acid molecule which encodes an amino acid sequence whichcomprises an amino acid sequence which has more than 90% identity to anamino acid sequence selected from the group consisting of: SEQ ID NO 2and SEQ ID NO 4, wherein said identity can be determined using theDNAsis computer program and default parameters;

(c) a nucleic acid molecule which is an allelic variant of a nucleicacid molecule selected from the group consisting of: a nucleic acidmolecule of (a); and a nucleic acid molecule of (b); and

(d) a nucleic acid molecule fully complementary to a nucleic acidmolecule selected from the group consisting of: a nucleic acid moleculeof (a); a nucleic acid molecule of (b); and a nucleic acid molecule of(c).

Also provided are vectors, in particular vectors comprising induciblepromoters, and for those comprising inducible promoters, in particularthose which are tightly regulated. Recombinant plant cells, seeds, plantparts, embryos and plants comprising these vectors are also provided.Preferred are plants selected from the group consisting of tobacco,cotton, corn, soybeans, and tomatoes.

Also provided by the present invention are isolated nucleic acidcompounds comprising a nucleic acid compound which hybridizes understringent conditions to a nucleic acid molecule selected from the groupconsisting of:

(a) SEQ ID NO 1 and SEQ ID NO 3.

Stringent hybridization conditions are determined based on definedphysical properties of the gene to which the nucleic acid molecule isbeing hybridized, and can be defined mathematically. Stringenthybridization conditions are those experimental parameters that allow anindividual skilled in the art to identify significant similarities-between heterologous nucleic acid molecules. These conditions are wellknown to those skilled in the art. See, for example, Sambrook, et al.,(1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LabsPress, and Meinkoth, et al., 138 Anal. Biochem. 267 (1984), each ofwhich is incorporated by reference herein in its entirety. As explainedin detail in the cited references, the determination of hybridizationconditions involves the manipulation of a set of variables including theionic strength (M, in moles/liter), the hybridization temperature (°C.), the concentration of nucleic acid helix destabilizing agents (suchas formainide), the average length of the shortest hybrid duplex (n),and the percent G+C composition of the fragment to which an unknownnucleic acid molecule is being hybridized. For nucleic acid molecules ofat least about 75 nucleotides or more, these variables are inserted intoa standard mathematical formula to calculate the melting temperature, orTm, of a given nucleic acid molecule. As defined in the formula below,Tm is the temperature at which two complementary nucleic acid moleculestrands will disassociate, assuming 100% complementarity between the twostrands: Tm=81.5° C.+16.6 log M+0.41(%G+C)−500/n−0.61(% formamide). Fornucleic acid molecules smaller than about 50 nucleotides, hybridstability is defined by the dissociation temperature (Td), which isdefined as the temperature at which 50% of the duplexes dissociate. Forthese smaller molecules, the stability at a standard ionic strength isdefined by the following equation: Td=4(G+C)+2(A+T). A temperature of 5°C. below Td is used to detect hybridization between perfectly matchedmolecules.

Hybridization reactions are often carried out by attaching the nucleicacid molecule to be hybridized to a solid support such as a membrane,and then hybridizing with a labeled nucleic acid molecule, typicallyreferred to as a probe, suspended in a hybridization solution. Examplesof commnon hybridization reaction techniques include, but are notlimited to, the well-known Southern and northern blotting procedures.Typically, the actual hybridization reaction is done under non-stringentconditions, i.e., at a lower temperature and/or a higher saltconcentration, and then high stringency is achieved by washing themembrane in a solution with a higher temperature and/or lower saltconcentration in order to achieve the desired stringency.

Also well known to those skilled in the art is how base-pair mismatch,i.e. differences between two nucleic acid molecules being compared,including non-complementarity of bases at a given location, and gaps dueto insertion or deletion of one or more bases at a given location oneither of the nucleic acid molecules being compared, will affect Tm orTd for nucleic acid molecules of different sizes. For example, Tmdecreases about 1° C. for each 1% of mismatched base-pairs for hybridsgreater than about 75 bp, and Td decreases about 5° C. for eachmismatched base-pair for hybrids below about 50 bp. Conditions forhybrids between about 50 and about 75 base-pairs can be determinedempirically and without undue experimentation using standard laboratoryprocedures well known to those skilled in the art. These simpleprocedures allow one skilled in the art to set the hybridizationconditions (by altering, for example, the salt concentration, theformamide concentration or the temperature) so that only nucleic acidhybrids with less than a specified % base-pair mismatch will hybridize.Stringent hybridization conditions are commonly understood by thoseskilled in the art to be those experimental conditions that will allowhybridization between molecules having about 30% or less base-pairmismatch (i.e., about 70% or greater identity). Because one skilled inthe art can easily determine whether a given nucleic acid molecule to betested is less than or greater than about 50 nucleotides and cantherefore choose the appropriate formula for determining hybridizationconditions, he or she can determine whether the nucleic acid moleculewill hybridize with a given gene under stringent hybridizationconditions and similarly whether the nucleic acid molecule willhybridized under conditions designed to allow a desired amount of basepair mismatch.

A preferred isolated nucleic acid compound of the present invention isone which comprises SEQ ID NO 3, with SEQ ID NO 1, or a fragment thereofbeing most preferred. SEQ ID NO 1 is a most preferred fragment of SEQ IDNO 3. However, an isolated nucleic acid compound which is anapproximately 0.9 kb cDNA fragment isolated from Microplitis croceipesteratocytes, and which fragment hybridizes to SEQ ID NO 1 or SEQ ID NO 3under stringent conditions, is preferred.

Included within the scope of the present invention, with particularregard to the nucleic acids above, are allelic variants, degeneratesequences and homologues. Allelic variants are well known to thoseskilled in the art and would be expected to be found within a giveninsect, plant or microbe and/or among a group of two or more insects,plants or microbes. The present invention also includes variants of TSPdue to laboratory manipulation, such as, but not limited to, variantsproduced during polymerase chain reaction amplification or site directedmutagenesis. It is also well known that there is a substantial amount ofredundancy in the various codons which code for specific amino acids.Moreover, variants of the universal code, such as are present in someplant, animal and fungal mitochondria, the bacterium Mycoplasmacapricolum [Yamo et al., 82 Proc. Natl. Acad. Sci. (USA) 2306 (1985)] orthe ciliate Macronucleus, may be used when the nucleic acid is expressedusing these organisms. Therefore, this invention is also directed tothose nucleic acid sequences which contain alternative codons which codefor the eventual translation of the amino acid. Methods to mutate genesare well-known to those in the art. Several books, including text booksand laboratory manuals are available on the subject. For instance,Sambrook et al., Molecular Cloning. A Laboratory Manual (Cold SpringHarbor Laboratory Press, 1989); Ausubel et al., Current Protocols inMolecular Biology (Greene Publishing Associates, Inc., 1993) aretypical. Moreover, commercial ventures sell kits that have instructionsand materials that even those with less than ordinary skill couldfollow. Kits can be obtained from http://www.sciquest.com, (800)233-1211.

Also included within the scope of this invention are mutations either inthe nucleic acid sequence or the translated protein which do notsubstantially alter the ultimate physical properties of the expressedprotein. For example, substitution of valine for leucine, arginine forlysine, or asparagine for glutamine may not cause a change infunctionality of the polypeptide. Lastly, a nucleic acid sequencehomologous to the exemplified nucleic acid compounds (or allelicvariants or degenerates thereof) will have at least 70%, preferably 80%,and most preferably 90% sequence homology with the nucleic acidcompounds in the sequence listing. Most preferred is a mRNA which iscomplementary to the DNA compounds in the sequence listing. In otherwords, a mRNA nucleic acid sequence homologous to a DNA nucleic acidsequence is characterized by the ability to hybridize to the exemplifiednucleic acid compounds (or allelic variants or degenerates thereof)under stringent conditions. Stringent hybridization conditions, andalterations of amino acids in a polypeptide are well known, and aredescribed, for example, in Sambrook et al., Molecular Cloning. ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1989).

A variety of procedures known in the art may be used to molecularlyclone the present nucleic acids. These methods include, but are notlimited to, complementation for function following the construction of agenomic DNA library in an appropriate vector system. Another method isto screen a genoric DNA library constructed in a bacteriophage orplasmid shuttle vector with a labeled oligonucleotide probe designedfrom the amino acid sequence of the gene. An additional method consistsof screening genomic DNA libraries constructed in a bacteriophage orplasmid shuttle vector with a partial DNA encoding the gene. Thispartial DNA is obtained by specific PCR amplification of the gene DNAfragments through the design of degenerate oligonucleotide primers fromthe amino acid sequence of the purified gene product or by using anothermember of the gene family as a probe. Sambrook et al., MolecularCloning. A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989)and Ausubel et al., Current Protocols in Molecular Biology (GreenePublishing Associates, Inc., 1993) describe these procedures.Alternatively, the nucleic acids can be prepared as exemplified herein.

When the nucleic acid is prepared or altered synthetically, advantagecan be taken of known codon preferences of the intended host where thenucleic acid is to be expressed. For example, although nucleic acidsequences of the present invention may be expressed in bothmonocotyledonous and dicotyledonous plant species, sequences can bemodified to account for the specific codon preferences and GC contentpreferences of monocotyledons or dicotyledons as these preferences havebeen shown to differ. Murray et al., 17 Nucl. Acids Res. 477 (1989).Thus, the maize preferred codon for a particular amino acid may bederived from known gene sequences from maize. Maize codon usage for 28genes from maize plants are listed in Table 4 of Murray et al., supra.

The cloned nucleic acids may be expressed through the methods describedin the examples or methods known in the art. The DNA can berecombinantly expressed by molecular cloning into an expression vectorcontaining a suitable promoter and other appropriate transcriptionregulatory elements, and transferred into prokaryotic or eukaryotic hostcells to produce recombinant gene product. Techniques for suchmanipulations are fully described in Sambrook, et al., supra. Expressionvectors can be used to express genes in a variety of hosts such asbacteria, bluegreen algae, plant cells, insect cells, fungal cells andanimal cells. Expression vectors may include, but are not limited to,cloning vectors, modified cloning vectors, specifically designedplasmids or viruses.

Vectors which comprise the nucleic acid compounds are within the scopeof the present invention, as are plants transformed with the abovenucleic acid compounds. Vectors may be obtained from various commercialsources, including Clontech Laboratories, Inc. (Palo Alto, Calif.),Stratagene (La Jolla, Calif.), Invitrogen (Carlsbad, Calif.), NewEngland Biolabs (Beverly, Mass.) and Promega (Madison, Wis.).

Preferred vectors are those which are capable of transferring thesequences disclosed herein into plant cells or plant parts. Expressionvectors are preferred, with expression vectors comprising an induciblepromoter operably linked to the nucleic acid compound being morepreferred. “Inducible” promoters typically direct expression of apolynucleotide in a specific tissue or may be otherwise under moreprecise environmental or developmental control. The most preferredvectors herein provided are expression vectors comprising atightly-regulated inducible promoter operably linked to the nucleic acidcompound.

Environmental conditions that may effect transcription by induciblepromoters include pathogen attack, anaerobic conditions or the presenceof light. Examples of inducible promoters are the Adhl promoter which isinducible by hypoxia or cold stress, the Hsp70 promoter which isinducible by heat stress, and the PPDK promoter which is inducible bylight. Examples of promoters under developmental control includepromoters that initiate transcription only, or preferentially, incertain tissues, such as leaves, roots, fruit, seeds or flowers. Anexemplary promoter is the anther specific promoter 5126 (U.S. Pat. Nos.5,689,049 and 5,689,051). The operation of a promoter may also varydepending on its location in the genome. Thus, an inducible promoter maybecome fully or partially constitutive in certain locations.

A number of promoters can be used in the practice of the invention. Thepromoters can be selected based on the desired outcome. For the purposesof the present invention, a wound-inducible promoter may be used in theconstructions of the invention. Such wound inducible promoters includepotato proteinase inhibitor (pin II) gene (Ryan, 28 Annu. Rev.Phytopath. 425 (1990); Duan et al., 14 Nature Biotech. 494 (1996); wun1and wun2, U.S. Pat. Ser. No. 5,428,148; win1 and win2 (Stanford et al.,215 Mol. Gen. Genet. 200 (1989); systemin (McGurl et al., 225 Science1570 (1992); WIPI (Rohmeier et al., 22 Plant Mol. Biol. 783 (1993);Eckelkamp et al., 323 FEBS Let. 73 (1993); MPI gene (Corderok et al., 6Plant J. 141 (1994) and references contained therein. This invention isappropriate for use in all crops potentially attacked by lepidopteranpests. These crops include cotton, maize, soybean, tomato and tobacco.

Promoters as described in the previous paragraphs are also preferred foruse in the present vectors. Chemically inducible promoters, such asPR-2b and PR-2d genes which are induced by salicylic acid, can either beconstructed de novo according to known techniques, or obtained fromvarious commercial sources, including those described above ,forobtaining vectors. Construction of vectors comprising promoters in framewith nucleic -acids is known in the art, and can be accomplishedaccording to Sambrook et al., Molecular Cloning. A Laboratory Manual(Cold Spring Harbor Laboratory Press, 1989) and Ausubel et al., CurrentProtocols in Molecular Biology (Greene Publishing Associates, Inc.,1993). A general method for the construction of any desired DNA sequenceis provided in Brown et al., [68 Methods in Enzymology 109 (1979)]. Alsoprovided are vectors comprising rice actin and rice ubiquitinconstitutive promoters and the nucleic acid compounds herein. Inaddition, there are other plant viral promoters which may be used toexpress genes of the present invention.

Also included in the present invention are recombinant plant cells,recombinant seeds, recombinant plant embryos and recombinant plantscomprising the vectors described herein. Seeds, embryos, plants or plantparts which recombinantly express the present amino acid compound(s) orcomprise herein-disclosed constructs are preferred embodiments of thepresent invention. Of course, those in the art recognize that any seed,embryo or plant transformed with the present constructs which are usefulfor producing plants for biomass are within the scope of the presentinvention.

Therefore, the present invention includes seeds, embryos, plants orplant parts which recombinantly express an isolated nucleic acidcompound, wherein said nucleic acid compound encodes an amino acidcompound selected from the group consisting of: SEQ ID NO 4; a fragmentof SEQ ID NO 4; an amino acid compound encoded by an allelic variant ofa nucleic acid compound encoding SEQ ID NO 4; and a fragment of an aminoacid compound encoded by an allelic variant of a nucleic acid compoundencoding SEQ ID NO 4. The present invention also provides seeds,embryos, plants or plant parts which recombinantly express isolatednucleic acid compounds comprising a nucleic acid compound whichhybridizes under stringent conditions to SEQ ID NO 3.

For example, the following seeds, embryos, plants or plant partstransformed with herein-disclosed nucleic acid constructs are consideredwithin the present invention: rice; soybean; cotton; maize; beet;tobacco; wheat; barley; poppy; rape; sunflower; alfalfa; sorghum; rose;carnation; gerbera; carrot; tomato; egg plant; lettuce; chicory; pepper;melon; cabbage; canola; banana; papaya; casava; fruit and nut trees;tulip; orchid and lilly. Particularly preferred are: tobacco, cotton,maize, soybeans and tomatoes.

Transformation of cells with the nucleic acid compounds of the presentinvention can be accomplished according to known procedures. Forexample, infective, vector-containing bacterial strains (such asAgrobacterium rhizogenes and Agrobacterium tumefaciens) may be used fortransformation. Zambryski, 43 Ann. Rev. Pl. PhysioL Pl. Mol. Biol. 465(1992). The following procedures are also well-known: Pollen-tubetransformation [Zhon-xun et al., 6 Plant Molec. Bio. 165 (1988)]; directtransformation of germinating seeds [Toepfer et al., 1 Plant Cell 133(1989)]; polyethylene glycol or electroporation transformation [Christouet al., 84 Proc. Nat. Acad. Sci. 3662 (1987)]; and biolistic processes[Yang & Cristou, Particle Bombardment Technology for Gene Transfer(1994)]. The transformed cells are also within the scope of the presentinvention.

The transformed cells may be induced to form transformed plants viaorganogenesis or embryogenesis, according to the procedures of DixonPlant Cell Culture: A Practical Approach (IRL Press, Oxford 1987).

In another aspect of the present invention, there is provided isolatedTSP protein molecules, wherein said TSP protein molecule comprises anamino acid sequence selected from the group consisting of:

(a) an amino acid sequence encoded by a nucleic acid molecule which hasmore than 70% identity to a nucleic acid molecule selected from thegroup consisting of: SEQ ID NO 1 and SEQ ID NO 3, wherein said identitycan be determined using the DNAsis computer program and defaultparameters; and

(b) an amino acid sequence which comprises an amino acid sequence whichhas more than 70% identity to an amino acid sequence selected from thegroup consisting of: SEQ ID NO 2 and SEQ ID NO 4, wherein said identitycan be determined using the DNAsis computer program and defaultparameters.

Recombinant plant cells and plants comprising the disclosed proteins arealso provided. Preferred are those recombinant plants selected from thegroup consisting of tobacco, cotton, corn, soybeans, and tomatoes.

Isolated TSP protein molecules with 65% to 100% sequence identity to thesequence listing compounds are provided. Indeed, those which are 75%,80%, 85%, and 95% identical are specifically pointed out as part of thepresent invention. Moreover, also provided are those isolated TSPprotein molecules, wherein said TSP protein molecule comprises an aminoacid sequence selected from the group consisting of:

(a) an amino acid sequence encoded by a nucleic acid molecule which hasmore than 90% identity to a nucleic acid molecule selected from thegroup consisting of: SEQ ID NO 1 and SEQ ID NO 3, wherein said identitycan be determined using the DNAsis computer program and defaultparameters; and

(b) an amino acid sequence which comprises an amino acid sequence whichhas more than 90% identity to an amino acid sequence selected from thegroup consisting of: SEQ ID NO 2 and SEQ ID NO 4, wherein said identitycan be determined using the DNAsis computer program and defaultparameters.

Recombinant plant cells and plants comprising the disclosed proteins arealso provided. Preferred are those recombinant plants selected from thegroup consisting of tobacco, cotton, corn, soybeans, and tomatoes.

Most preferred is an isolated amino acid compound which is SEQ ID NO 2,although any portion of SEQ ID NO 2 which has the ability to causeimpaired growth and development in lepidoptera larvae is considered partof the present invention as well. Modifications of the amino acidcompounds, such as conservative changes in the amino acid sequence,removal of proteolytic cleavage sites to stabilize the peptide, ormodifications useful to identify existence or location of gene productsare also within the scope of the present invention. For example, anengineered antibody recognition site would be helpful for researchpurposes, or for quality-control in a commercial plant. Suchmodifications can be accomplished according to Young et al., [9 Mol.Plant-Microb. Interact. 105 (1994)].

The amino acid compounds of the present invention can be purifiedaccording to common purification techniques, such as that described inBollag et al., Protein Methods (Wiley-Liss 1996); Scopes, ProteinPurification: Principles and Practice (Springer-Verlag 1994); Doonan,Protein Purification Protocols (Humana Press 1996). For example,artisans will also recognize that these compounds (or portions thereof)can be synthesized by well-known solid phase peptide synthesis orrecombinant DNA methods.

For purification via recombinant DNA methods, following expression ofthe nucleic acids disclosed above in a recombinant host cell, the aminoacid compounds described herein can be recovered in purified form.Several purification procedures are available and suitable for use. Forexample, the amino acid compounds may be purified from cell lysates andextracts by various combinations of, or individual application of saltfractionation, ion exchange chromatography, size exclusionchromatography, hydroxyapatite adsorption chromatography and hydrophobicinteraction chromatography. In addition, the amino acid compound can beseparated from other cellular proteins by use of an immunoaffinitycolumn made with monoclonal or polyclonal antibodies specific for thefull length nascent gene product, or polypeptide fragments of the geneproduct. Finally, if the vector is modified to include a poly-histidinetag, the expressed protein can be purified by nickel affinitychromatography.

Also provided are methods to construct an insect resistant plant,comprising: introducing into a plurality of plant cells an isolatednucleic acid compound selected from the group consisting of:

(a) a nucleic acid molecule which has more than 70% identity to anucleic acid molecule selected from the group consisting of: SEQ ID NO 1and SEQ ID NO 3, wherein said identity can be determined using theDNAsis computer program and default parameters

(b) a nucleic acid molecule which encodes an amino acid sequence whichcomprises an amino acid sequence which has more than 70% identity to anamino acid sequence selected from the group consisting of: SEQ ID NO 2and SEQ ID NO 4, wherein said identity can be determined using theDNAsis computer program and default parameters; and

(c) a nucleic acid molecule which is an allelic variant of a nucleicacid molecule selected from the group consisting of: a nucleic acidmolecule of (a); and a nucleic acid molecule of (b); and causing atleast some of the plant cells to grow into at least one plant; andselecting those plants which contain the nucleic acid introduced.

Therefore, also provided are formulations for topical pesticidescomprising the amino acid compounds herein disclosed. For instance TSP(SEQ ID NO 2) can be used as an ingredient, or alone, in a mixture,suspension, etc. for application directly to Heliothis-infested orpotentially infested plants. Alternatively, the TSP gene (either SEQ ID1 or SEQ ID 3 or modifications thereof) might be used with a recombinantbaculovirus insecticide whereby the baculovirus infects the insect andas the virus reproduces it over expresses the TSP gene. Bonning andHammock, 41 Ann. Rev. Entomol. 191 (1996).

In particular, there are provided methods to confer insect resistanceproperties to a plant, comprising applying to the surface of a plant anisolated nucleic acid molecule selected from the group consisting of:

(a) a nucleic acid molecule which has more than 70% identity to anucleic acid molecule selected from the group consisting of: SEQ ID NO 1and SEQ ID NO 3, wherein said identity can be determined using theDNAsis computer program and default parameters

(b) a nucleic acid molecule which encodes an amino acid sequence whichcomprises an amino acid sequence which has more than 70% identity to anamino acid sequence selected from the group consisting of: SEQ ID NO 2and SEQ ID NO 4, wherein said identity can be determined using theDNAsis computer program and default parameters; and

(c) a nucleic acid molecule which is an allelic variant of a nucleicacid molecule selected from the group consisting of: a nucleic acidmolecule of (a); and a nucleic acid molecule of (b).

Also in particular, there are provided methods to confer insectresistance properties to a plant, comprising applying to the surface ofa plant an isolated amino acid molecule selected from the groupconsisting of:

(a) an amino acid sequence encoded by a nucleic acid molecule which hasmore than 70% identity to a nucleic acid molecule selected from thegroup consisting of: SEQ ID NO 1 and SEQ ID NO 3, wherein said identitycan be determined using the DNAsis computer program and defaultparameters; and

(b) an amino acid sequence which comprises an amino acid sequence whichhas more than 70% identity to an amino acid sequence selected from thegroup consisting of: SEQ ID NO 2 and SEQ ID NO 4, wherein said identitycan be determined using the DNAsis computer program and defaultparameters.

Moreover, there are provided methods to confer constitutive and/orinducible Lepidoptera resistance to a plant, comprising transforming theplant with a vector comprising the nucleic acid sequences describedherein. Since Lepidoptera resistance is one characteristic conferred toa plant by the TSP genes disclosed herein, an ideal method would be toactivate the sequences disclosed herein for the amount of time and inquantities necessary to fight the pest, and no longer, since the genesutilize energy that could otherwise be used to create biomass and/orfruit development.

In particular, there are provided methods to confer Lepidopteraresistance in a plant, comprising: transforming at least one plant witha inducible promoter-comprising vector of the present invention, andintroducing to the plant an inducing agent capable of inducing theinducible promoter of the vector, and providing the time and conditionsneeded to cause inducement.

Also provided are methods to induce a Lepidoptera resistance response ina plant transformed with the nucleic acids described herein, comprising:introducing to the plant an agent or environmental conditions capable ofinducing the inducible promoter of the vector, and providing the timeand conditions needed to cause inducement, and causing inducement.Preferred agents are pathogens, and preferred environmental conditionsare wounds. However, it is also within the scope of the presentinvention to select a plant part into which a vector is transformed, andselectively cause Lepidoptera resistance in response to a developmentalstage of the plant, such as upon flowering or at a particular fruitdevelopmental stage. For instance, Heliothis larvae prefer feeding inthe growing bud and developing fruit. Resistance could be engineered tocoincide with these growing tissues.

Transformation of plants with these sequences would be according towell-known procedures as described above. Plants can be grown accordingto well-known procedures.

EXAMPLES Example 1—Insect Populations

Heliothis virescens larvae were reared on artificial diet in acontinuous colony maintained at the 25±2° C. with a 16:8 (L:D)photoperiod at the University of Kentucky as described by Vanderzant [55J. Econ. Entomol. 140 (1962)]. Prospective host Heliothis larvae wereremoved from the colony as pharate fourth instars and parasitized forboth colony maintenance and subsequent collection of parasite eggs.Parasitization was accomplished by exposing host larvae forapproximately 1 h to mated female M. croceipes wasps at a ratio of 8:1.Parasitized larvae were placed into individual cups containing diet andheld at 27±2° C. After emergence, the adult parasite wasps were kept atan approximate 1:1 sex ratio and supplied with honey:water (1:1). Zhanget al., 20 Arch. Insect Biochem. Physiol. 231 (1992).

Example 2—General Procedures

The in vitro culturing of teratocytes and subsequent collection of theproteins secreted by the teratocytes were described by Schepers et al.[44 J. Insect Physiol. 767 (1998)]. Host H. verescens larvae weresuperparasitized by placing 10 larvae and an equal number of wasps in a15×1.5 cm plastic petri dish which was placed in a lighted incubator at27° C. for 2 h. Individual superparasitized larvae were placed in 18 mLplastic cups containing diet and held at 27° C. for 28 h. M. croceipeseggs were obtained by sterile dissection of superparasitized H.verescens larvae 28 h after parasitization. Following surfacesterilization by emersion in 95% and 70% ethanol, the larvae were heldin sterile water until used for dissection. A larva was placed in a wellof a depression slide along with 100 μL of Ex-cell 400 medium (JHRBiosciences, Lexana, Kans.) with 60 μg/mL of gentamicin sulfate. Twofine forceps were used to open the integument without breaking themidgut. The exposed midgut was dipped into the medium and the integumentwas massaged with forceps to force the M. croceipes eggs into themedium. Eggs were collected with a capillary micropipette and washedfive times by transfer to fresh sterile 100 μL drops of Ex-cell 400. Tenwashed eggs were placed in each of three 100 μL drops of Ex-Cell 400placed in a 5×0.9 cm tightly sealed Petri dish. The dishes were held inan incubator at 27° C. The eggs hatch approximately 14 h afterdissection with each egg yielding approximately 900 teratocytes. Zhanget al. 23 Int. J. Insect Morphol. & Embryol. 173 (1994). The teratocytesdissociate from the chorion and begin secreting proteins (TSP). For TSPcollection the teratocytes, parasite larvae and medium were placed intomicrocentrifuge tubes, the parasite larvae were allowed to settle, thenthe medium and teratocytes were removed with a pipette to a separatemicrocentrifuge tube which was centrifuged for 5 min at 800×g. Thesupernatant contained the crude TSP. One larval equivalent (LE) of TSPis that amount (˜2 μg of protein) secreted by an in vitro culture ofteratocytes derived from a larva during a period of three days. In somecases, the fraction of TSP that passes through a 30 kD molecular weightfilter but is retained by a 3 kD filter was used for assays (Schepers etal. supra). The dye-binding Bio-Rad protein reagent (Bio-RadLaboratories, Hercules, Calif.) was used to determine proteinconcentrations using bovine serum albumen as a standard assay [Bradford,72 Anal. Biochem. 248 (1976)]. Centricon (Amicon, Inc., Beverly, Mass.)molecular weight cutoff filters were used following the manufacture'sdirections. SDS-PAGE was performed using a standard method [Laemelli,227 Nature 680 (1970)] and visualized with Coomassie blue and/or silverstain. Southern and northern blots followed standard procedures[Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold SpringHarbor Laboratory Press (1989); Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates, Inc., (1993)]. Reversedphase HPLC using a 2.1×250 mm VyDac C18 column, 5μ, 300A packingmaterial was used to separate protein components in the 3-30 kD fractionand to separate peptide fractions after hydrolysis of the isolated 13.9kD protein. The flow rate was 150 μL/min with a binary gradient changingthe percentages of 0.06% TFA versus 0.054% TFA in 70% acetonitrile overa 2 h period. Each HPLC peak was collected, concentrated, and subjectedto SDS-PAGE.

Example 3—Expression of the 14 kD cDNA

The 13.9 kD protein was purified from approximately 1500 LE of TSPobtained from teratocytes cultured in vitro. Details related to the useof specific procedures cited in this example are described Example2—General Procedures. The protein of interest passed through a 30 kDmolecular weight filter but was retained by a 3 kD filter. Schepers etal., 44 J. Insect Physiol. 767 (1998). The 13.9 kD protein was separatedfrom the other 3 TSP proteins in the biologically active fraction byreversed phase HPLC. The most abundant protein (13.9 kD) was selectedfor the initial analysis. It was digested with the Lys-C protease andrechromatographed on a C 18 column. Two of the fragments were selectedfor amino acid sequencing using an Applied Biosystems 477A ProteinSequencer. One fragment provided a sequence of only 5 amino acids(VTWYN) while the other fragment was sequenced through 28 positions(PFDFSDDGNOSCAPASGICHRVGLEITK).

To select a cDNA encoding TSP, 2 degenerate primers were synthesizedtaking advantage of inferred lysine residues N-terminal to the derivedpeptide sequence (inferred because of Lys-C digestion); terat 1 (SEQ IDNO 5)=AARGTNACNTGGTAYAA; and terat 2 (SEQ ID NO 6)=AARCAYCCNTTYGAYTT.The 3′ end and subsequently the 5′ end of the cDNA was isolated usingthe PCR based approach of rapid amplification of cDNA ends [RACE,Frohman IN: Innis PC et al., PCR Protocols, Academic Press 28 (1990);Clackson et al. IN: McPherson et al., PCR: A Practical Approach, IRLPress, 187, (1991)]. Briefly, an oligo-dT primer (ODT) was used tosynthesize cDNA from 1 μg of teratocyte RNA. The degenerateoligonucleotide primers, terat 1 and terat 2, were used in parallelreactions to prime second strand cDNA synthesis then amplified in aconventional PCR reaction (terat 1+ODT and terat 2+ODT). The terat 1+ODTreaction produced a single major amplimer of about 150 bp while theterat 2+ODT reaction produced a major product of approximately 540 bp.The 540 bp amplimer was cloned and sequenced. The signal peptide anduntranslated leader sequence were obtained from a 5′ RACE product. Thecomposite sequence encodes all 33 amino acids known from peptidesequence data and shows that the terat 2 peptide sequence is N-terminalto the terat 1 peptide, as suggested by the initial amplificationproducts. The complete cDNA contains an open reading frame of 426nucleotides that encodes a 23 amino acid signal peptide and a total of129 amino acids with a predicted molecular weight of 14,012. After theremoval of the signal peptide, the predicted weight of the secretedprotein is 11,466, relatively close to the 13.9 kD molecular weight ofthe most abundant protein in the 3-30 kD fraction, as estimated bySDS-PAGE. The “inferred” lysine residue used in construction of theterat 2 oligonucleotide was not present in the predicted peptidesequence because this peptide sequence was from the N-terminus of themature protein. A 17 bp primer-specified poly A tail was preceded by twoconsensus poly-adenylation signals.

Inspection of the predicted amino acid sequence from the cDNA encodingthe 14 kD TSP revealed a cysteine-motif similar to those previouslydescribed from a polydnavirus (PDV). Alignment of the TSP-cysteine motifwith the 6 motifs from Campoletis sonorensis PDV shows that all 6cysteine residues were conserved at similar spacing to those observed inCsPDVs. A 4 amino acid core (KPCC) was also present in the TSP motif.

The cDNA encoding TSP was expressed only from teratocytes, was encodedby the wasp genome, apparently as a single copy gene, and did nothybridize to the M. croceipes PDV genome (T. Schepers and Webb,unpublished). To localize the TSP gene, DNA was extracted from adultmale M. croceipes wasps and from viral DNA. Two micrograms of viral DNAand 10 μg of male wasp genomic DNA was blotted to nylon membrane in adot blot apparatus. Cloned TSP was used as a positive control with pZerovector DNA used as a negative control (5 μg each). Only the wasp genomicand positive control DNAs hybridized to the TSP probe. Viral and malewasp genomic DNA were then digested with BamH1 and Xba1 and hybridizedunder high stringency conditions (50% formamide, 5×SSC, 5×Denhardt's 20mM Na phosphate with 100 μg/mL sheared salmon sperm DNA at 42° C.) tothe TSP probe after electrophoresis on a 0.7% agarose gel and transferto nylon membrane. Two TSP hybridizing bands were visible on the genomicSouthern blot suggesting that the TSP gene exists as a single copy genein M. croceipes chromosomal DNA. The 14 kD cDNA was expressed as afusion to a poly-histidine tag that simplifies protein purification inboth prokaryotic (pET vectors, Novagen) and eukaryotic systems(baculovirus expression system) (BaculoGold, PharMingen). Webb andSummers, 87 Proc. Nat'l Acad. Sci. 4961 (1990). An appropriate portionof the amino acid sequence of TSP was synthesized and linked to keyholelympet hemocyanin and used for antibody preparation.

Southern blots. The TSP cDNA was used to probe DNA prepared from thewasp teratocytes and from M. croceipes polydnavirus (McPDV). The TSPcDNA bound exclusively to DNA from the wasp teratocytes.

Molecular analysis of the TSP cDNA. The protein expressed from thebaculovirus system was purified for functional analyses. Three differentpreparations of recombinant TSP were prepared and assessed with thetestes assay. A 0.5 μg/100 μL dose of TSP resulted in a 27.1±2.2% (n=11)reduction in protein synthesis by testes. Based on this information andthe assumed molecular weight of 11,466, the concentration would beapproximately 0.436 μmolar. Three LE of crude TSP used in a parallel setof experiments conducted at the same time yielded a 31.0±6.0% (n=9)reduction in protein synthesis. The control testis of each pair receivedan equivalent volume of buffer that was identical to that obtained fromthe TSP preparation.

Example 4—Immunological Analyses

To produce a TSP antiserum, a peptide region of the TSP predicted tohave a high degree of antigenicity was synthesized according to themethods described in Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory (1988) and linked to keyhole lympethemocyanin. Polyclonal antibodies were raised following immunizationprocedures described by Huebers et al. [158 J. Comp. Physiol. B 291(1988)]. The synthesized peptide was injected into rabbits with anadjuvant. Four weeks after the initial immunization the rabbits wereboosted with a similar amount of protein. Two weeks after the primaryboost, the antiserum was collected and evaluated with western blotscontaining the crude TSP and hemoloymph from parasitized insects. Boostimmunizations were performed until an antiserum with suitablespecificity and titer was produced. Antiserum binding, specificity andreactivity were analyzed by transferring proteins from 16.5% SDS-PAGEgels to Immobilion-P (Milipore; Bedford, Mass.) membranes following theprocedure of Li and Webb [68 J. Virol. 7482 (1994)] with alkalinephosphatase-conjugated anti-rabbit immunoglobulin G and4-bromo-4-chloro-3-indolylphosphate p-toluidine salt and nitrobluetetrazolium chloride as chromogenic substrates. In some trials, in orderto achieve greater sensitivity, enhanced chemiluminescence withhorseradish peroxidase and luminol substrate was used following theinstructions provided with the kit (Pierce Chemical Co., Rockford,Ill.).

An immune response was evident to the bacterial TSP, but no protein wasdetected in either the TSP fractions or in purified TSP from therecombinant virus. However, the antiserum obtained from a rabbitinjected with a synthesized region of TSP and linked to keyhole lympethemocyanin reacted to the protein that has been identified as thesecreted TSP. The antiserum will be used for a variety of studiesincluding the localization and function of TSP in host larval tissuesand localization of TSP in transgenic plants.

Example 5—Testis Assay

Pairs of testes removed from digging Day 2 (D2) 5th stadium H. verescenswere processed in a manner similar to the fat body organ culturedescribed by Schepers et al. [44 J. Insect Physiol. 767 (1998)]. Asingle testis of the pair was placed in a total volume of either 50 or100 μL of methionine depleted medium and a volume of test material.Protein synthesis was measured by [35S]-methionine incorporation duringa 4 h incubation. The ratio of cpm from one of the testes pair incubatedwith the treatment (TSP or candidate fraction) and the other one of thetestes pair (control) was used to measure reduction in proteinsynthesis.

In an assay that used 3 LE of crude TSP/50 μL of medium, we found asignificant decrease (82.8±5.4%) in the incorporation of[35S]-methionine in the treated testes compared to the controls (n=25).Schepers et al. supra reported that a significant amount of the activityassociated with reduction in protein synthesis in fat body organ assayswas confined to the fraction of TSP that passed through a 30 kD filterbut was retained by a 3 kD filter. When this fraction was assessed usinga rabbit reticulocyte lysate assay with mRNA from H. verescens testes, adose of 3.5 LE/50 μL caused a 51.3±1.4 % decrease in the incorporationof [35S]-methionine (n=3). Although the percentage reduction was lesswith the concentrated fraction of TSP, it is likely that not all theactive component was retained with the fraction, some being lost duringthe purification process.

Example 6—In Vitro Translation Assays

The effect of TSP on mRNA translation was assessed using a rabbitreticulocyte translation system (Life Technologies, Gaithersburg, Md.).One μg of mRNA was used in a total volume of 25 μL. TSP and buffercontrols were concentrated with 3 kD Amicon filters prior to addition tothe assay mixture. A wheat germ extract translation system (Promega,Madison, Wis.) also was used to assess the activity of 0.5 μg of TSP ina plant-derived system.

When the recombinant TSP was tested with a rabbit reticulocyte lysateassay an inverse dose-dependent effect was observed over a range of0.2-0.8 μg protein/25μL (FIG. 1). The concentration of TSP at thehighest dose would be approximately 2.79 μmolar.

The wheat germ assay is similar to the rabbit reticulocyte translationsystem except that wheat germ tissue is used as a source of enzymes,cofactors, etc. It was used to evaluate the effect of TSP mRNAtranslation in a cell-free system using plant-derived components. In asingle experiment with six replications 0.5 μg of TSP significantlyreduced [35S]-methionine uptake by 25%.

Example 7—In Vivo Developmental Impairment

Qualitative and quantitative changes in hemolymph proteins in H.verescens were observed in larvae injected with either M. croceipesteratocytes or crude TSP. Hemolymph protein titers in hosts receivingeither 0.5 or 1 LE of teratoctyes were similar to those of parasitizedlarvae, whereas a single injection of 4 LE of crude TSP was required toinduce a similar response. Treated larvae required several days longerthan controls to reach a comparable premetamorphic stage[burrowing-digging according to Webb and Dahlman, 2 Arch. InsectBiochem. Physiol. 131 (1985)]. Reductions in fat body proliferationsimilar to those seen in parasitized larvae were observed in larvaetreated with either 1 LE of teratocytes, or with 2 or 4 LEs of crudeTSP. Proliferation of perivisceral fat body weights from larvae treatedwith either teratocytes or crude TSP was significantly reduced, in adose dependent manner, when compared to controls. Both light- andtransmission-electron microscopy observations revealed cytologicaldifferences in fat body tissues of larvae injected with eitherteratocytes or crude TSP from the condition observed in parasitizedlarvae and noninjected controls. Gross dissection of perivisceral fatbody from parasitized, teratocyte-injected and TSP-injected larvaeshowed tissue much less developed and differed considerably inappearance from controls. Observed differences included reduced sizeand/or number of lipid bodies and qualitative and quantitative changesin other cytoplasmic organelles [Zhang et al., 43 J. Insect Physiol. 577(1997)].

Example 8—Expression of Teratocyte Secretory Protein (TSP) Gene inTransgenic Plants Renders Resistance to Insect Feeding

Experimental Protocol:

Plant and enzymes: Tobacco plants (Nicotiana tabacum CV Samsun NN) wereused for plant transformation. Transgenic tobacco seeds (Rl and R2progeny) were germinated in the presence of Kanamycin (220 μg/mL) forselecting transformed seedlings. Restriction enzymes, DNA modifyingenzymes, and DNA and RNA isolation kits were purchased from commercialsources from Gibco-BRL Life Technologies (Rockville, Md.) and usedaccording to the manufactures' specifications. Nitrocellulose membranesfor hybridization analysis were obtained from Schleicher & Schuell(Keene, N.H.).

Construction of plant expression vectors pK117, pK118 and pK119: Theplasmid pVL1392-TSPhis containing the cDNA gene of teratocyte secretoryprotein (TSP) from Microplitis croceipes, a member of the Braconidaewasp family, was used as starting genetic material. The DNA fragmentscorresponding to the coding sequence of TSP with and without its signalpeptide (TSPdSP) were isolated by PCR amplification from the plasmidpVL1392-TSPhis. For PCR reaction, to isolate TSP with its signalpeptide, the following primers were used: a 39-mer forward primer,(named 5′TSP#88) (SEQ ID NO 7),5′-d(GCGGGCTCGAGACCATGGGTCCATCCAAAATTTTAATT)-3′ with 18nt complementaryto the 5′ end of the TSP gene coding region (amino acid coordinates #2to 7 of TSP), and XhoI and NcoI restriction cleavage sites (underlined)and translation initiation codon (bold); a 42-mer reverse primer, (named3′TSP#90) (SEQ ID NO 10)5′-d(ATGCAGGGGCTCTTAGGTCGACCCGGGCCCTTTTTTCTTGTA)-3′ with 18 ntcomplementary to the 3′ end of the TSP gene (amino acid coordinates #126to 131 of TSP), flanked with two restriction sites SalI and SstI and astop codon (bold). These restriction sites were introduced into the PCRamplified TSP gene fragment for cloning facilities in plant expressionvectors.

For PCR amplification of the TSP gene without its signal peptide a39-mer forward primer: (named 5′TSPdSP#89) (SEQ ID NO 8),5′-d(GCGGGCTCGAG AACCATGGGT CATCCATTCGATTTTTCT)-3′ with 18 ntcomplementary to the 5′ end of the TSP gene (amino acid coordinates #23to 28 of TSP), and XhoI and NcoI sites (underlined) and a translationinitiation codon (bold); and the reverse primer, 3′TSP#90 describedabove were used. The PCR amplification was carried out for 30 cyclesunder the following standard condition: denaturation (92° C. for 1 min),annealing (55° C. for 1 min), synthesis (72° C. for 2 min). The PCRgenerated TSP gene fragment, with and without the signal peptide, weredigested with XhoI and SstI, gel purified and cloned into thecorresponding sites of pBS (KS+) for DNA sequencing. The resultingplasmids were named pBT and pBdT, respectively. Before use, all PCRproducts were sequenced by dideoxy chain terminator method [Sanger etal., 74 Proc. Nat'l. Acad. Sci. USA 5463 (1997)] using a syntheticprimer. The TSP fragments with and without the signal peptide wereisolated after restriction digestion of pBT and pBdT respectively withNcoI and SstI, the fragments were gel purified and cloned into thecorresponding sites of pBS-AlMV5′ [Maiti et al., 90 Proc. Nat'l Acad.Sci. USA 6110 (1993)] which contains the 5′ut7 untranslated region ofAlMV RNA 4. The resulting plasmids were designated as pB5′T and pB5′dTrespectively, have the general structure:5′-XhoI-AlMV-RNA4-5′untranslated region-NcoI-TSP genefragment-SalI-SstI-3′. The XhoI-SstI fragment of TSP with and withoutthe signal peptide was cloned separately into the corresponding sites ofthe expression vector pKLP36 [Maiti and Shepherd, 244 Biochem. Biophys.Res. Comm. 440 (1998); Maiti and Shepherd, U.S. Pat. No., 5,850,019(1998)]. The TSP gene is under the control of a modified PC1SVfull-length transcript promoter. The resulting expression vectors weredesignated as pKT117 (which contains TSP with the signal peptide) andpKT118 which contains TSP without the signal peptide; see FIG. 2).

Construction ofpKT119: The coding sequence (corresponding to amino acidcoordinates 23 to 131) of TSP without its signal peptide was isolated byPCR amplification using pVL1392-TSPhis as a template. In PCR reactionthe appropriately designed following primers were used. The forwardprimer (5′TSP#140) (SEQ ID NO 9),5′-d(GCGGGCTCGAGAACCATGGGTGGATCCCATCCATTCGATTTTTCT)-3′ with 18 ntcomplementary to the 5′ end (italic) of the TSP gene (amino acidcoordinates #23 to 28 of TSP gene), along with XhoI, NcoI and BamHIsites (underlined) and a translation initiation codon (bold); and the42-mer reverse primer 3′TSP#90 described above were used. The PCRamplified fragment was gel purified, digested with XhoI and SstI andcloned into the corresponding sites of pBS(KS+). The resulting plasmidwas designated as pBdT2. The NcoI to SstI fragment containing TSPwithout the signal peptide was restricted from pBdT2, gel purified andcloned into the corresponding sites of pBS-AlMV5′ [Maiti et al., 90Proc. Nat'l Acad. Sci. USA 6110 (1993)] in order to incorporate the5′ut7 region of AlMV RNA4 as described above. The resulting plasmid wasnamed as pB5′dT2. A 30 amino acid long transit sequence from PR1b genewas isolated from pBSAPM (a plasmid containing PR 1b transit sequence)by PCR amplification using appropriately designed primers to insert NcoIsites at the 5′ end, and BamHI site at the 3′ end. The PCR product fortransit peptide PR1b was gel purified and digested with NcoI and BamHIand inserted into the corresponding sites of pB5′dT2. The Xhol to SstIfragment from the resulting plasmid pB5′RdT2 was cloned into thecorresponding sites of plant expression vector pKLP36 (Maiti andShepherd supra, supra). The resulting plant expression vector wasdesignated as pKT119.

Plant transformation and analysis of transgenic plants: The plantexpression vectors were introduced into Agrobacterium tumefaciens strainC58C1:pGV3850 by triparental mating and tobacco (Nicotiana tabacum CVSamsun NN) was transformed with the engineered Agrobacterium asdescribed earlier [Maiti et al., 90 Proc. Nat'l Acad. Sci. USA 6110(1993)].

PCR analysis: The integration of TSP gene in the genome of transgenicplants (R1 and R2 progeny) was detected by PCR amplification usingappropriately designed primers specific for TSP or TSPASP (TSP withoutsignal peptide) gene sequence. The specificity of each PCR product wasanalyzed by Southern hybridization with a TSP probe.

RNA isolation, RNA dot blot and Northern analysis: Total RNA wasisolated from the transgenic tobacco seedlings (R2 progeny) developedwith pKT117 and pKT118 and untransformed control seedlings (Samsun NN).Transformed seedlings developed with pKLP36GUS [Maiti and Shepherd, 244Biochem. Biophys. Res. Comm. 440 (1998)] was used as a vector control.Total RNA was isolated with guanidine thiocyanate solution as describedearlier [Maiti et al., 57 Virus Research 113 (1998)]. Proceduresfollowed for RNA dot blot and Northern analysis have been described indetails (Dey and Maiti, 40 Plant Mol. Biology, 71 (1999).

Western blot analysis: Protein was extracted from leaves of transformedplants with TSP constructs and transformed plants with GUS construct asvector control, and nontransformed control plants. Leaf tissue washomogenized in extraction buffer (0.0625M Tris-HCl pH6.8, 10% glycerol,2% SDS, 10% 2-mercaptoethanol), boiled for 5 min, and centrifuged at12000×g for 10 min. Total soluble leaf proteins (25 μg) were separatedon a 12.5% SDS-PAGE, transferred to nitrocellulose, and probed with theantiserum raised against the synthetic peptide of TSP. Blots weredeveloped with chemiluminescence reagents from a Pierce kit describedabove.

Results from the Molecular Analysis of Transgenic Plants:

The chimeric TSP gene with and without its signal peptide wereintroduced into tobacco plants by Agrobacterium-mediated transformationas described earlier [Maiti et al., 90 Proc. Nat'l Acad. Sci. USA 6110(1993)]. The coding sequence of TSP gene was placed under the control ofmodified PC1SV FLt promoter and rbcs E9 terminating sequence in theplant expression vector pKLP36 [Maiti and Shepherd, U.S. Pat. No.5,850,019 (1998)]. The 5′ ends of the TSP genes were fused to theuntranslated leader sequence of AlMV RNA 4 sequence to enhance thetranslation of mRNA. The upstream PC1SV FLt promoter was modified withthe double enhancer domain in order to stimulate the transcription ofchimeric gene in plants [Maiti and Shepherd, U.S. Pat. No. 5,850,019(1998)]. Transgenic plants derived from each transforment were assayedfor the integration and expression of genes by PCR and RT-PCR RNA dotblot and Northern blot and immunobloting analysis. Transgene were stablyintegrated as shown by PCR analysis for R1 and R2 progeny seedlings(data not shown). Accumulation of TSP mRNA in leaf of transgenic plantswere evaluated by RNA dot blot and Northern analysis (FIG. 3). TheNorthern analysis of total RNA isolated from homozygous lines (R2progeny/3rd generation) showed the expected size of TSP and TSPΔSPtranscripts (FIG. 3).

Proper Mendelian segregation of the KanR marker gene was observed.Homozygous tobacco independent lines (based on KanR marker gene)expressing TSP and TSPΔSP were taken for insect-bioassay.

Example 9—Insecticidal Activity of Transgenic Tobacco Leaf Tissue

Insect bioassays were performed on homologous transgenic lines (R2progeny) transgenic tobacco plants transformed with the constructspKT117 and pKT118 containing the TSP with and without its signalpeptide, respectively, were tested for insect tolerance by feedingexcised leaves to neonate lepidopteran larvae, tobacco budworm (H.verescens) and tobacco hornworm (Manduca sexta).

The plants to be tested were germinated from seeds on nutrient agarplates, then transferred to nutrient agar placed in the bottom ofsterile 6×6×10 cm tall plastic boxes where they were grown underartificial light for approximately four weeks. By this time the plantshas attained heights of approximately 10 cm. Leaf material was harvestedfrom these plants, as needed, to supply fresh tissue to each insect inthe bioassay.

The assay arena consisted of a 10×50 mm tightly sealed plastic petridish containing a 4.25 cm disk of Whatman #1 filter paper, moistenedwith 100 μL of water. Initially, an approximately 2.5 cm² portion ofleaf tissue was removed from the plant and placed on the moist filterpaper. To each dish was added a single neonate H. verescens larvae. Tenlarvae were used for each treatment. On the third day after the test wasinitiated, each larva was observed under a dissecting binocularmicroscope and the developmental stadium was recorded as either beingactively feeding or in the premolt or pharate stage of the next stadium.Additional leaf material was added as needed. The width of the headcapsule shed after each larval ecdysis was measured at the widest point.Beginning on the fifth day of the test, each larvae was weighed to thenearest 0.1 mg. Also on the fifth day, the contents of the dish andfilter paper was replaced. This time the paper was moistened with 75 μLof water because the mass of leaf tissue added had increased to thepoint that less supplemental water was required to maintain leafturgidity. The test was terminated on the seventh day, all larvae werefrozen and the ultimate head capsule widths subsequently determined.

A total of 22 transformed tobacco lines have been tested; 11 containingthe TSP gene with the signal peptide and 11 containing the TSP genewithout the signal peptide. Three different sets of controls wereusually included with each group of transformed plants that were tested.These were untransformed tobacco, cultivar Samsun NN; plants transformedwith pKLP36GUS, a gene with no known insecticidal activity [Maiti andShepherd, 244 Biochem. Biophys Res. Comm. 440 (1998)]; and artificialdiet.

Three different parameters were used to assess insecticidal activity.1). Weighted values for time to specific developmental stages for days3, 4, 5, 6 and 7 of the experiment. Even in standardized assays, not alllarvae feed and grow at the same rate. Therefore, each developmentalstage (instar) was assigned a numerical score as a useful way tostatistically evaluate delayed development of H. verescens larvae. Thefollowing values were assigned: 2nd instar=1, premolt 2nd instar=2, 3rdinstar=3, premolt 3rd instar=4, 4th in premolt 4th instar=6, 5thinstar=7. 2). Mean weight of larvae on days 5, 6 and 7 of theexperiment. 3). Mean width of head capsule (mm) for each instar. Thedata was analyzed with a one-way AOV and LSD (T) pairwise comparisons ofmeans using a Statistix 4.1 program.

Of the 22 transformed lines tested, six yielded significantly lowervalues than the GUS control with at least one of the three parametersmeasured. An additional four lines showed trends that may or may not bestatistically different from GUS controls. Table 2 shows that after aseven day feeding trial with selected lines the average developmentalstage was approximately one rank less than those feeding on the GUStransformed control. The mean weight gained by larvae in these tests hadsubstantial standard error and only one of the transformed lines yieldedsignificantly reduced weight gain. In three of the lines, head capsulewidths were significantly smaller than controls in at least someinstars, an indication of less conversion of ingested food to body mass.Four of the six most promising lines were transformed without the signalpeptide. However, one of the transformed lines with the signal peptideappears to be one of the better of the group.

Because of limited plant material, only a few tests with larvae ofManduca sexta were conducted. The test arena was that same used for H.verescens but because neonate M. sexta larvae are much larger than H.verescens they consume much more leaf material. It appeared that M.sexta was much more sensitive to the TSP gene expressed in thetransformed plants than H. verescens (Table 3). Lines containing the TSPgene without the signal peptide were the most effective. Even though thenumber of tests was very small, mortality, as well as inhibition ofgrowth and development, was evident within five days after placement onthe transformed plant material.

Conclusion

In conclusion, the cDNA encoding TSP, when purified from baculovirusexpression systems, inhibits protein synthesis in organ cultures, rabbitreticulocyte lysate assays and wheat germ extract assays. Inhibition oftranslation was similar to that observed when the 30-kD TSP fraction wasassayed. In vivo studies showed that injections of crude TSP causedinhibition of growth, developmental arrest and various physiologicalchanges similar to those caused by injections of teratocytes orparasitization by the M. croceipes wasp.

The TSP gene, with and without the signal peptide, has been expressed intransgenic tobacco plants. Heliothis larvae offered some lines of thesetransgenic tobacco plants grew and developed more slowly than controls.Manduca larvae also grew more slowly and experienced higher mortalitythan larvae fed control plants.

Although the present invention has been fully described herein, it is tobe noted that various changes and modifications are apparent to thoseskilled in the art. Such changes and modifications are to be understoodas included within the scope of the present invention as defined by theappended claims.

TABLE 1 Sequence listing brief description SEQ ID NO Description ofSequence 1 TSP cDNA sequence (without signal) 2 TSP amino acid sequence(without signal) 3 TSP cDNA sequence (with signal) 4 TSP amino acidsequence (with signal) 5 Terat 1 6 Terat 2 7 5′TSP#88 8 5′TSPdSP#89 95′TSP#140 10 3′STP#90

TABLE 2 Response of tobacco budworm to transgenic plants expressing TSPgene with and without its signal peptide. Response of neonate H.virescens larvae after feeding seven days on transgenic tobacco plantslines. Weighted Stage^(a) Mean Weight % Weight Plant Line ± S.E. LSD^(b)± S.E. (mg) LSD Reduction GUS (Control) 5.9 ± 0.3 A 78.2 ± 10.5 A 0pKLP36GUS(3)R1#2 7311 5.3 ± 0.2 AB 68.0 ± 8.5  A 13 117(3)R1#3 71019 4.8± 0.6 B 55.6 ± 22.8 A 28.9 117(10)R1#9 8115 5.0 ± 0.0 B 59.5 ± 10.0 A23.9 118(1)R1#5 8615 4.6 ± 0.8 B 47.3 ± 21.1 B 39.5 118(6)R1#5 8711 5.2± 0.2 AB 74.9 ± 10.8 A 4.2 118(7)R1#1 8816 5.1 ± 0.4 B 69.8 ± 11.8 A11.8 118(8)R1#6 ^(a)Each developmental stage assigned a numerical valuewith larger values indicating greater developmental rate. ^(b)LSD =Least Significant Difference analysis of mean separation. Valuesfollowed by the same letter are not significantly different (P ≦ 0.05).

TABLE 3 Response of tobacco hornworm to transgenic plants expressing TSPgene with and without its signal peptide. Response of neonate M. sextalarvae after feeding five days on transgenic tobacco plant lines. MeanWeight Plant Line ± S.E. (mg) LSD^(a) % Mortality at 5 Days GUS(Control) 31.8 ± 5.2  A 0 pKLP36GUS(3)R1#3 7311 29.4 ± 11.1 AB 0117(3)R1#1 8115 12.6 ± 2.5  BC 50 118(1)R1#5 8711 9.7 ± 3.3 C 25118(7)R1#1 8816 8.4 ± 1.3 C 50 118(8)R1#6 ^(a)LSD = Least SignificantDifference analysis of mean separation. Values followed by the sameletter are not significantly different (P ≦ 0.05).

10 1 320 DNA Microplitis sp. 1 ccattcgatt tttctgatga tggaaatcaaagctgtgctc cggcttcagg aatctgccat 60 cgagtaggat tagaaattac caaaccgtgttgtaataaat tcgatcgttg tttcgcttca 120 gtatctgaac ccgtgtctcg ttgtggtgggacggattact cggtagcagt tgtaacagtt 180 ctttcgattg taccaaagtt cagggtgcaacttgtgaaaa cgggatatgt acttgcggaa 240 aagatgctac tgagtacaca agacacagatgtaaaccaaa tcacatgtcc ccgaaagtta 300 catggtacaa caaaaaatga 320 2 106 PRTMicroplitis sp. 2 Pro Phe Asp Phe Ser Asp Asp Gly Asn Gln Ser Cys AlaPro Ala Ser 1 5 10 15 Gly Ile Cys His Arg Val Gly Leu Glu Ile Thr LysPro Cys Cys Asn 20 25 30 Lys Phe Asp Arg Cys Phe Ala Ser Val Ser Glu ProVal Ser Arg Cys 35 40 45 Gly Gly Asp Gly Leu Leu Gly Ser Ser Cys Asn SerSer Phe Asp Cys 50 55 60 Thr Lys Val Gln Gly Ala Thr Cys Glu Asn Gly IleCys Thr Cys Gly 65 70 75 80 Lys Asp Ala Thr Glu Tyr Thr Arg His Arg CysLys Pro Asn His Met 85 90 95 Ser Pro Lys Val Thr Trp Tyr Asn Lys Lys 100105 3 389 DNA Microplitis sp. 3 atgccatcca aaattttaat ttcactcggaatatttctaa ctatttatgt tagttatata 60 tccgctcatc cattcgattt ttctgatgatggaaatcaaa gctgtgctcc ggcttcagga 120 atctgccatc gagtaggatt agaaattaccaaaccgtgtt gtaataaatt cgatcgttgt 180 ttcgcttcag tatctgaacc cgtgtctcgttgtggtggga cggattactc ggtagcagtt 240 gtaacagttc tttcgattgt accaaagttcagggtgcaac ttgtgaaaac gggatatgta 300 cttgcggaaa agatgctact gagtacacaagacacagatg taaaccaaat cacatgtccc 360 cgaaagttac atggtacaac aaaaaatga 3894 129 PRT Microplitis sp. 4 Met Pro Ser Lys Ile Leu Ile Ser Leu Gly IlePhe Leu Thr Ile Tyr 1 5 10 15 Val Ser Tyr Ile Ser Ala His Pro Phe AspPhe Ser Asp Asp Gly Asn 20 25 30 Gln Ser Cys Ala Pro Ala Ser Gly Ile CysHis Arg Val Gly Leu Glu 35 40 45 Ile Thr Lys Pro Cys Cys Asn Lys Phe AspArg Cys Phe Ala Ser Val 50 55 60 Ser Glu Pro Val Ser Arg Cys Gly Gly AspGly Leu Leu Gly Ser Ser 65 70 75 80 Cys Asn Ser Ser Phe Asp Cys Thr LysVal Gln Gly Ala Thr Cys Glu 85 90 95 Asn Gly Ile Cys Thr Cys Gly Lys AspAla Thr Glu Tyr Thr Arg His 100 105 110 Arg Cys Lys Pro Asn His Met SerPro Lys Val Thr Trp Tyr Asn Lys 115 120 125 Lys 5 17 PRT Microplitis sp.UNSURE (6)..(9) X = unspecified or any amino acid 5 Ala Ala Arg Gly ThrXaa Ala Cys Xaa Thr Gly Gly Thr Ala Tyr Ala 1 5 10 15 Ala 6 17 PRTMicroplitis sp. UNSURE (9)..(9) X = unspecified or any amino acid 6 AlaAla Arg Cys Ala Tyr Cys Cys Xaa Thr Thr Tyr Gly Ala Tyr Thr 1 5 10 15Thr 7 38 DNA Microplitis sp. 7 gcgggctcga gaccatgggt ccatccaaaa ttttaatt38 8 39 DNA Microplitis sp. 8 gcgggctcga gaaccatggg tcatccattc gatttttct39 9 45 DNA Microplitis sp. 9 gcgggctcga gaaccatggg tggatcccatccattcgatt tttct 45 10 42 DNA Microplitis sp. 10 atgcaggggc tcttaggtcgacccgggccc ttttttcttg ta 42

We claim:
 1. An isolated teratocyte secretory protein (TSP) nucleicacid, wherein said nucleic acid is selected from the group consistingof: (a) a nucleic acid selected from the group consisting of: SEQ ID NO1; and SEQ ID NO 3, and (b) a nucleic acid molecule fully complementaryto a nucleic acid molecule selected from the group consisting of: SEQ IDNO 1; and SEQ ID NO
 3. 2. A vector comprising a nucleic acid of claim 1.3. A vector of claim 2, wherein said vector further comprises aninducible promoter operably linked to said nucleic acid.
 4. A vector ofclaim 3, wherein said inducible promoter is tightly regulated.
 5. Arecombinant plant cell comprising a vector of claim
 3. 6. A recombinantseed comprising a vector of claim
 3. 7. A recombinant plant embryocomprising a vector of claim
 3. 8. A recombinant plant comprising avector of claim
 3. 9. A recombinant plant of claim 8, wherein said plantis selected from the group consisting of tobacco, cotton, corn,soybeans, and tomatoes.